Infection with viral pathogens represents a significant source of morbidity and mortality in human populations, and thus a public health threat for which we must prepare. It is well appreciated that infection across the mucosal surfaces represents the primary means by which pathogens initiate infection in the human host. Therefore, defining the critical immune pathways which catalyze protective immunity at the mucosal level represents an important endeavor in the fight against infectious diseases, with pertinent clinical implications. In this study, we have employed vaginal infection of mice with herpes simplex virus type 2 (HSV-2) as a model system to identify the cellular and molecular pathways which provide protective innate mucosal immunity. Our groups, and others, have recently demonstrated that vaginal delivery of several different Toll-like receptor (TLR) agonists protects mice against otherwise lethal HSV-2 disease, by stimulating local innate immunity. However, the precise pathways responsible for such protection remain largely unexplored. Recently, we demonstrated that plasmacytoid dendritic cells (pDCs) are required for CpG-mediated protective immunity, and that pDC-stimulated protection occurs under conditions in which mucosal epithelial cells cannot respond to secreted IFNs, suggesting the existence of an IFN-independent protective pathway. Furthermore, pDCs are also critical for protective immunity during natural HSV-2 infection. The purpose of this proposal is to define the IFN-independent mechanisms by which pDCs provide protective innate immunity at the mucosal surface. Here, we will test the requirement of several candidate effector molecules in pDC-dependent protective immunity such as TNF Related Apoptosis-Inducing Ligand (TRAIL), a known mediator of pDC lytic activity, as well as FasL, TNFR, granzyme, and perforin, both in vitro and in vivo. Such mechanistic insights have important implications in pDC-dependent antiviral immunity and may suggest novel treatments for numerous mucosal viral infections. Viral infections, and specifically mucosal virus infections, are a leading cause of morbidity and mortality in the human population. The work proposed here seeks to identify the mechanisms by which the immune system protects from such infections as well as to develop antiviral treatments with increased efficacy. Thus, these studies harbor promising potential to help curtail the harmful effects of virus-induced disease and, in turn, improve the quality of life for millions of individuals